L’inibizione di MEK potenzia l’effetto antitumorale del Triossido di Arsenico in cellule di Leucemia Mieloide Cronica Bcr-abl+ resistenti all’Imatinib: studio preclinico in vitro ed in vivo.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of the Philadelphia chromosome, which generates the constitutively active kinase Bcr-Abl of 210 Kd (p210). Imatinib (Gleevec) is a small molecule tyrosine kinase inhibitor (TKI) that effectively targets the Bcr-Abl kinase. Imatinib mediates remission in the majority of patients with CML, but patients can develop resistance most frequently through Bcr-Abl overexpression or acquired point mutations in the kinase domain of Bcr-Abl that block Imatinib binding to Bcr-Abl. In most patients, resistance against Imatinib occurs from Bcr-Abl point mutations. Second-generation TKIs, such as Nilotinib and Dasatinib, have shown efficacy in the treatment of patients with many of the Bcr-Abl mutations, except for T315I mutation, completely resistant to second-generation TKIs. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. The aim of this study was to investigate whether the combined treatment with highly selective inhibitors of MEK phosphorylation (PD 184352 or PD0325901) and arsenic trioxide (ATO) has cytotoxic effects on cell lines expressing various Bcr-Abl alterations, including T315I mutation. We found that treatment with PD strongly enhanced the cytotoxic effect of ATO in all tested CML cell lines, and combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in the majority of cell lines tested, including T315I+ cell line. Immunoblotting analyses demonstrated that treatment with PD inhibited the ATO-mediated up-regulation of Bcr-Abl and CrkL activation, a recognised substrate of Bcr-Abl. Therefore, we studied whether the p53-related gene p73 is molecular target of the combined treatment in T315I+ cells. We found that the combination PD+ATO promoted the accumulation of the proapoptotic and antiproliferative TAp73 protein (PD-mediated) and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔNp73 protein (ATO-mediated) thus elevating TA/ΔNp73 ratio that was significantly higher than either treatment alone (3 fold increase respect to PD and 1.5-fold increase respect to ATO). Consistent with these results, we found that PD+ATO increased expression of p73 target gene p53AIP1(p53-regulated apoptosis-inducing protein 1) and loss of mitochondrial depolarization. To determine the biologic relevance of p73 family proteins in response to PD+ATO treatment, we silenced the expression of endogenous TAp73 and ΔNp73 transcripts by using specific siRNA. We found that the selective down-regulation of TAp73 strongly decreased the PD+ATO induced-apoptosis, whereas functional knock-out of ΔNp73 significantly enhanced PD+ATO cytotoxicity, thereby confirming the key role of these proteins in PD+ATO-induced apoptosis. Finally, we studied PD+ATO combination in a clinically relevant mouse model of Imatinib-resistant BCR-ABL-dependent disease. Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were injected intravenously with Bcr-Abl-T315I-expressing cell line. Mice with Bcr-Abl-T315I-induced Imatinib resistant leukemia developed aggressive disease, typically resulting in death in 30 days. Histopathological, immunohistochemical and molecular analysis of 20 days Imatinib-treated mice revealed massive infiltration and increased size of liver and spleen. In contrast, spleens and livers from PD+ATO-treated mice demonstrated substantially normal tissue architecture. Moreover PD+ATO-treated mice showed significantly prolonged survival compared to Imatinib, confirming the PD+ATO-triggered inhibition of leukemic progression. Our preclinical in vitro and in vivo studies suggest that PD+ATO-treatment could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl+ leukemia, including those harbouring the T315I mutation.